Vascular dysfunction and vascular injury, as manifested by impairments in microvascular perfusion, autoregulation, and vascular reactivity, as well as by vasogenic edema, hemorrhage, inflammation, and coagulation, contribute importantly to outcome following focal stroke. Treatment strategies may derive from studies of "ischemic tolerance", wherein endogenous mechanisms of protection are activated by different "preconditioning" stimuli. Studies outlined in this proposal will focus on elucidating the innate mechanisms responsible for protection of cerebrovascular endothelium following hypoxic preconditioning, utilizing a mouse model of induced tolerance to focal stroke and a human cerebrovascular endothelial cell culture model of ischemic tolerance, both developed and characterized in our laboratory. Key induction and expression mechanisms whereby hypoxic preconditioning attenuates diverse endpoints of ischemia-induced vascular/endothelial dysfunction and injury will be elucidated in each model. Specific Aim 1: Elucidate the role of endothelial nitric oxide synthase (eNOS) in the induction and expression pathways responsible for hypoxic preconditioning-induced cerebrovascular ischemic tolerance in vivo and in vitro. We hypothesize that eNOS-derived NO is integral to the signaling pathways responsible for inducing the transcription/translation of vasculoprotective genes. We also hypothesize that eNOS is an effector protein contributing to the ischemia-tolerant phenotype of the preconditioned cerebral microcirculation. Specific Aim 2: Delineate the involvement of the transcription factor HIF-2alpha in mediating cytoprotective gene expression in endothelial cells in vivo and in vitro in response to hypoxic and chemical preconditioning. We will test the hypothesis that the activity of HIF-2alpha, highly expressed in endothelial cells, is upregulated by these preconditioning stimuli, is modulated by eNOS-derived NO and phosphorylated Akt, and is critical to mediating the expression of cytoprotective genes in endothelium that lead to ischemic tolerance. Specific Aim 3: Elucidate the participation of survivin, a specific inhibitor-of-apoptosis family member, in the resistance of in vivo- and in vitro-preconditioned endothelium to ischemic injury, and the mechanisms by which this protein affords protection. We hypothesize that hypoxic preconditioning upregulates survivin expression, which blocks caspase-mediated endothelial injury, thereby preserving post-ischemic microcirculatory homeostasis. Identification of endogenous mechanisms of endothelial cell resistance to ischemic injury may provide novel molecular targets for the therapeutic treatment of vascular dysfunction and vascular injury following focal stroke.